A wide variety of applications in basic biomedical and clinical research and in molecular medicine require specific detection of one or more nucleic acids (e.g., studies of gene expression). Levels of RNA expression, for example, have traditionally been measured using Northern blot and nuclease protection assays. However, these approaches are time-consuming and have limited sensitivity, and the data generated are more qualitative than quantitative in nature. A multiplex screening assay for mRNA was recently reported that combines nuclease protection with luminescent array detection (Martel et al. (2002) “Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection” Assay Drug Dev Technol. 1:61-71). However, although this assay has the advantage of measuring mRNA transcripts directly from cell lysates, limited assay sensitivity and reproducibility were reported.
Another exemplary technique, the real-time or quantitative polymerase chain reaction (qPCR), has been gaining widespread use in quantification of nucleic acid since its development (Higuchi et al. (1993) “Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions” Biotechnology 11:1026-1030). This is primarily due to qPCR's excellent detection sensitivity and broad dynamic range, simple homogeneous assay format, and quantitative capability. However, the quantitative capability of the qPCR assay for mRNA is significantly compromised by the pre-analytical steps of RNA isolation and conversion to cDNA, which result in significant drops in assay reproducibility and accuracy (see, e.g., Bustin (2000) “Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays” Journal of Molecular Endocrinology 25:169-193, Bustin (2002) “Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): Trends and problems” J Mol Endocrinol 29:23-39, and Bustin and Nolan (2004) “Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction” J Biomol Tech. 15:155-66). There are several reasons why the pre-analytical steps potentially result in qPCR assay variations. First, isolated RNA can be of variable quality and stability. Second, the efficiency of conversion of RNA to cDNA is dependent on many factors, including reverse transcription enzyme efficiency, the presence of inhibitors in the reverse transcription reaction, template abundance, the presence of background nucleic acids, and different reverse transcription priming methods. Third, extensive degradation and modification of RNA can occur in samples such as formalin-fixed paraffin embedded tissue, which substantially affect the efficiency of RNA isolation from those samples and the quality of conversion of the isolated RNA to cDNA. Because of the need for RNA isolation and cDNA conversion in the current qPCR format, additional issues arise such as genomic DNA contamination, 3′ bias, presence of inhibitors in the PCR reaction, interference by other cDNAs within the nucleic acid mixture, amplification efficiency variation among different samples, mispriming, and primer-dimer formation, among others.
Furthermore, the current qPCR format is based on target amplification, and, as a result, target-specific primers and probes need to be designed and validated for every target analyzed. Because the primers and probes are designed using a single gene sequence of limited genetic complexity, yet the PCR is conducted in the presence of a complex cDNA mixture, usually several primer and probe pairs need to be designed and validated in order to select a primer and probe pair with close to 100% amplification efficiency and no primer-dimer formation. In a multiplex qPCR format, mutual interference of multiple sets of PCR primers and probes can exacerbate the primer and probe selection problem, substantially increasing the amount of work required for assay design and validation. Also, a qPCR reaction for quantification of a particular target nucleic acid usually requires upfront optimization in primer and probe concentration, in Mg2+ and dNTP concentrations, and in hot-start PCR to achieve highest amplification efficiency and to prevent mispriming and primer-dimer formation. Finally, assay reproducibility is particularly problematic when working with very low copy numbers of target nucleic acids because of stochastic effects. Particle distribution statistics predict that a greater number of replicates is required to differentiate five from 10 copies of a target molecule than to differentiate 500 from 1000 copies.
Therefore, there is a significant need to develop a qPCR and/or other nucleic acid detection method that eliminates the steps of RNA isolation and reverse transcription. There is also a significant need to develop a nucleic acid detection and/or quantification method that does not involve amplification of the target nucleic acid. Among other aspects, the present invention provides methods that overcome the above noted limitations and that permit rapid, simple, and sensitive detection and/or quantitation of nucleic acids. A complete understanding of the invention will be obtained upon review of the following.